Xenograft soft tissue implants and methods of making

ABSTRACT

The present application is directed to the field of implants comprising soft tissue for use in implantation in humans. The soft tissue implants of the present application are preferably obtained from xenograft sources. The present application provides a chemical process that sterilizes, removes antigens from and/or strengthens xenograft implants. The present techniques yield soft tissue implants having superior structural, mechanical, and/or biochemical integrity. The present application is also directed to processes for treating xenograft implants comprising soft tissues such as dermis, and to implants produced by such processes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/839,440, filed Mar. 15, 2013; which is a Continuation-in-Part of PCTApplication No. PCT/EP2012/000124, filed Jan. 12, 2012 which claimspriority from German application DE102011008604.8, filed Jan. 14, 2011,the disclosures of which are incorporated by reference therein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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TECHNICAL FIELD

The present application is directed to the field of biological tissueimplants and biological tissue implant processing for transplantation,preferably into humans. The tissue implants are preferably xenograftsoft tissue implants, although certain aspects of the presentapplication may apply to other tissues from allograft or xenograftsources. The present application provides a chemical process thatsterilizes, removes antigens from and/or strengthens xenograft implants.The present techniques yield soft tissue implants having desiredstructural and mechanical properties, and/or biochemical integrity. Thepresent application is also directed to processes for treating xenograftimplants comprising soft tissues such as dermis, and to implantsproduced by such processes.

BACKGROUND OF THE INVENTION

Many injuries and ailments throughout the human body are treated throughsurgical intervention utilizing either biological tissue or syntheticmaterial implants. Among these are conditions including spinaldegeneration, sports medicine, podiatric, trauma and general orthopedicinjuries or maladies involving bone or hard tissue. Also commonlybenefiting from surgical intervention utilizing either biological tissueor synthetic material implants are soft tissue conditions includinghernia, urological, gynecological, cardiac, neural, and generalabdominal injuries or maladies.

In selecting implants to address these various injuries and maladies,surgeons are often faced with tradeoffs between natural, biologicaltissue materials and synthetic materials.

Biological materials often offer natural healing, incorporation andregenerative capability, but may be lacking in material properties orhandling characteristics as compared to synthetics. Surgeons musttherefore often choose to accept the lack of regeneration andincorporation potential in using a synthetic material, which is at bestinert and at worst inflammatory and prone to infection, in order to findan implant with the physical handling and material properties requiredfor a particular surgical application.

Donated human cadaver (i.e. allograft) tissue has proven to be anefficacious option allowing patients to return to their pre-injuryquality of life. However, allograft options are burdened with thechallenge of depending on a raw material that has large variability andlimited availability. Surgeons have tried to mitigate graft variabilityby limiting donor criteria (e.g. only accepting grafts from donors <45years of age); however, such limitations have also exacerbated theavailability challenge.

Thus, a goal is to find a sufficient pool of donor tissue, with similargenetic, physical and physiological attributes, that could mitigate oreliminate the above problems. This can be done by utilizing xenografttissue. With a xenograft tissue source donor genetic makeup can beselected and controlled through breeding and animal management,production can be monitored and controlled to ensure the best health andmuscle tone, and donor age can be planned and selected. Such donorswould thus have exceptional biomechanical structures available forcreating high quality grafts which would be recovered from them at theirpeak age.

Implants comprising soft tissues may be implanted into a recipient toreplace and/or repair existing soft tissues. For example, hereditarydefects, disease, and/or trauma may damage soft tissues such thatreplacement and/or repair is desirable. These implants may beallografts, autografts, or xenografts, and the recipients may be human,mammal, or animal recipients. Implants are frequently used where therecipient is a human patient. Implants comprising soft tissues have beenused, including in human patients, to replace heart valves, ligaments,tendons and skin, among other tissues.

It is desirable to treat implants, particularly autografts, allografts,and xenografts, to remove, inactivate or substantially reduce one ormore undesirable components or to instill one or more desirablecomponents. For example, implants may be passivated, or treated toremove or inactivate bacteria, viruses, fungi and other pathogens andantigenic constituents.

A desirable treatment process includes one or more of the followingfeatures: decellularization, effective removal or inactivation of a widerange of bacterial, viral and fungal pathogens; absence of grafttoxicity; retention or improvement of desirable tissue characteristics,such as biomechanical strength or growth-inducing properties;effectiveness across a wide range of operating modifications and/or fora wide variety of tissue types; ability to conclude the process in afinal implant tissue container, to ensure sterile packaging and deliveryfor implantation; ability to apply automated control and monitoringsystems and develop an automated and validated process.

A desirable treatment process for xenograft dermal tissue also removesor substantially reduces hair and hair follicles.

BRIEF SUMMARY OF THE INVENTION

The present application is directed to a method of processing axenograft dermal tissue implant to make it more suitable fortransplantation into a human. In one embodiment, the method comprisessplitting the dermal tissue, treating the dermal tissue to remove hair,exposing the dermal tissue to a saline solution, exposing the dermaltissue to an oxidizing sterilant; and dehydrating the dermal tissue.

The present application is also directed to a method of processing axenograft implant where the implant is made from dermal tissue ofporcine origin.

The present application is also specifically directed to embodimentsutilizing oxidizing sterilants such as peroxides, oxides, hypochlorites,percarboxylic acids, and ozone. Hydrogen peroxide (H₂0₂) is preferred.

The present application is also specifically directed to embodimentsthat expose the dermal tissue to a saline solution in multiple steps.Specifically, one embodiment includes multiple steps of exposing thedermal tissue to a saline solution, with steps of exposing to water inbetween. This process creates an osmotic gradient.

The present application is also specifically directed to embodimentsutilizing sodium sulfide to remove hair from the xenograft dermaltissue.

The present application is also specifically directed to embodimentsthat further include a rehydration step that occurs after thedehydration step.

The present application is also specifically directed to embodimentsthat further include punching or stamping grafts of a desired shape fromthe dermal tissue.

The present application is directed to a method for the preparation of agraft from animal dermis. In one embodiment, the method comprisesproviding an animal dermis; treating the animal dermis with an aqueoussodium sulfide solution, once or several times; treating the dermis withan aqueous salt solution, once or several times; treating the dermiswith an aqueous hydrogen peroxide solution; and dehydrating the dermis.In specific embodiments, the animal dermis is porcine dermis. Thepresent application is also directed to grafts made by the aboveprocess.

The present application is also directed to embodiments that furtherinclude using an aqueous alkaline, sodium sulfide-containing solution.In specific embodiments, an aqueous solution is used which contains from0.01 to 10 weight-% of sodium sulfide. In other specific embodiments, anaqueous solution is used which in addition to sodium sulfide contains0.001 to 0.5 M sodium hydroxide. In further specific embodiments, thedermis is treated in an aqueous sodium sulfide containing solution for 1to 48 hours, with a solution volume in milliliters that is 5 to 15 timesthe mass of the dermis in grams.

The present application is also directed to embodiments that treat thedermis two to ten times with an aqueous, 1 to 50 wt-% salt solution,wherein the salt is an alkali metal or alkaline earth metal, such assodium chloride, potassium chloride, lithium chloride, magnesiumchloride or calcium chloride.

The present application is also directed to embodiments that treat thedermis two to five times with an aqueous solution containing 1 to 10wt-% of hydrogen peroxide, with optional rinsing between each treatment.

The present application is also directed to embodiments that dehydratethe dermis by treating from two to ten times with an organic solventselected from the group consisting of acetone, methanol, ethanol andisopropyl alcohol.

The present application is also directed to embodiments that include inproviding the dermis several steps such as: cleaning the skin ofslaughtered pigs with detergent-containing water at a temperature of notmore than about 40° C.; removing the skin from the back portion of thepig; removing the bristles from the skin provided above and removal ofthe subcutaneous tissue and the epidermis from the dermis using asplitting machine.

The present application is also directed to a method of surgerycomprising implantation of a graft prepared by the methods above inhernia surgery, for the treatment of hernia or abdominal repair.

These and other advantages and novel features of the presentapplication, as well as details of illustrated embodiments thereof willbe more fully understood from the following description of the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the regions of porcine tissue for evaluation.

FIGS. 2A and 2B show a histological examination of processed porcinedermal inferior regions. FIG. 2A shows H&E stained tissue with 50×magnification and frozen cryostat sectioning. FIG. 2B shows Alcian Bluestained tissue with 100× magnification and frozen cryostat sectioning.

The foregoing summary, as well as the following detailed description ofcertain embodiments of the present application, will be betterunderstood when read in conjunction with the appended drawings. For thepurposes of illustration, certain embodiments are shown in the drawings.It should be understood, however, that the claims are not limited to thearrangements and instrumentality shown in the attached drawings.Furthermore, the appearance shown in the drawings is one of manyornamental appearances that can be employed to achieve the statedfunctions of the system.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present application is directed to the field ofbiological tissue implants and biological tissue implant processing fortransplantation. In a preferred embodiment, the present application isdirected to an acellular porcine dermis graft that maintains appropriatemechanical properties, for example, for surgical hernia repair. Theterms “graft” and “implant” are used interchangeably herein.

The goal is to sufficiently decellularize porcine dermis to create anacellular graft suitable for human implantation. In porcine dermissamples, this is difficult due to the matrix density and hair follicledepth inherent to this tissue. The process of this application achievesthis goal and produces tissue to be used as a graft material for humans.Such tissue must have certain handling characteristics, appropriatehistology, and strong biomechanics. The process of this applicationdecellularizes the porcine dermal matrix and maintains sufficientmechanical properties. One procedure where these grafts could beutilized is in human abdominal surgery. Thus, desirable mechanicalproperties were selected in part from literature review of humanabdominal wall biomechanics, where the highest load at failure of theabdominal wall tissue was 10±3.4 N/mm² and 39N [Hollinsky].

As used herein, the term “passivate” is intended to refer to theelimination of potentially pathogenic organisms and immunogenicsubstances from an implant. Thus, both sterility and reducedantigenicity is intended by this term, although elimination ofbeneficial biological properties of the implant, such as osteogenicproperties (osteoconduction or osteoinduction; bone fusion), naturaltissue functionality, and desirable structural strength of an implantare not intended by this term. The term “passivation” is preferred tothe term “sterilize” because, while sterilization is a goal, that termhas an absolute connotation for which the ability to definitively testis limited by the state of the art of making such measurements and/or bythe need for attendant tissue destruction. In addition, while theimplants produced according to the method of this application may not becompletely devoid of any antigenicity or pyrogenicity, these undesirableaspects are greatly reduced, and this too is intended by the term“passivation,” as used herein. To be suitable for implantation inhumans, the grafts (implants) of the present application must be treatedto remove, inactivate or substantially reduce any antigenic proteins,which may generate a rejection of the implant. It also must be treatedto remove, inactivate or substantially reduce any bacteria and viruses.A desirable treatment process for xenograft dermal tissue also removesor substantially reduces hair and hair follicles.

As used herein, the term “decellularize” is intended to refer to aprocess that eliminates or sufficiently reduces native cells andcellular material in a tissue such that, when such tissue is implanted,it does not invoke an adverse immune response. The term “acellular” isintended to refer to tissue that is sufficiently reduced in cells andcellular material as to not invoke an adverse immune response.

“Soft tissue”, as used herein, refers to any biological tissue otherthan bone, including but not limited to tendons, ligaments, fascia,whole joints, dura, skin (dermis), pericardia, heart valves, veins,neural tissue, submucosal tissue (e.g. intestinal tissue), andcartilage, or a combination thereof. The “soft tissue” described hereinis typically a collagenous material that is autograft, allograft orxenograft, is preferably xenograft and preferably dermis.

“Graft” (or “implant”), as used herein, refers to any material theimplantation of which into a human or an animal is considered to bebeneficial.

The present application is directed to processes for making a softtissue implant more suitable for implantation into a recipient,specifically by decellularization of the tissue. Soft tissues (such asdermis) treated according to the present techniques are wholly orpartially passivated by contact with cleaning agents, such as solutionscontaining any of an oxidizing sterilant (for example, hydrogenperoxide), saline, dehydrating agents and/or combinations thereof. Thetissues are preferably obtained from xenograft sources for implantationinto humans.

The grafts obtained after treatment are biocompatible in humanrecipients, with no substantively deleterious immune response orrejection due to xenogeneic antigens. The grafts obtained aftertreatment are in some embodiments bioactive, and achieve healing,remodeling and incorporation in the humans to which they are implanted.These grafts allow for neovascularization and reincorporation with thepatient's own tissue. Neovascularization is the proliferation of bloodvessels, usually in tissue not normally containing them, and/or of adifferent kind than usual in a tissue; neovascularization specificallycan include formation of functional microvascular networks with redblood cell perfusion. In the context of tissue grafts,neovascularization can include the formation of blood vessels within thetissue graft.

The method of this application provides for implant processing wherebybone marrow, blood, proteins (especially including hair), andparticulate matter is efficiently removed, such that what remains isessentially a tissue matrix, passivated tissue matrix, bioinert tissuematrix or bioactive tissue matrix in which a drastic reduction in anyform of endogenous material and/or viable organisms is achieved.Creating an acellular matrix is preferred.

In particular, it is noted that the processing methods of the presentapplication related to removal or reduction of antigenic material fromtissue may be practiced upon and may be useful for the production ofxenografts of multiple tissue types, including soft tissue grafts suchas tendons, ligaments, skin (dermis or dermal tissue), dura matter,fascia, and other connective tissues. In some embodiments dermal tissue(skin) is preferred.

The present processes comprise a novel sequence of cleaning agents fortreatment of xenograft soft tissue, preferably dermis. In preferredembodiments of the present processes, one or more of the cleaning agentsare contacted with the implant comprising the soft tissue to removeblood, fat, cells, proteins, antigens, bacterial, viral, fungal or othermaterial. Cleaning agents include, but are not limited to, detergents,disinfectants (sometimes called disinfecting agents), decontaminants(sometimes called decontaminating agents), antibiotics, virucidalcompounds, dehydrating agents and the like. Specialized agents can beused to enhance tissue properties and/or for tissue protection. One,non-limiting, example of a specialized agent are agents to removehair/hair follicles from dermal tissue. Additionally, solutions that arehypertonic or hypotonic can be used to establish osmotic gradients, forexample, by hypo/hyperosmotic soaks.

One embodiment, specifically developed for treating xenograft dermaltissue (e.g. of porcine origin), involves hair removal,hypo/hyperosmotic soaks, hydrogen peroxide, solvent dehydration, andoven drying.

The cleaning agents may be provided in the form of solutions or othermixtures. In some embodiments, the cleaning agent is provided as anaqueous solution (preferably using DI water). Also DI water itself canbe used a washing step or in creating osmotic gradients between steps.

For xenograft dermal tissue, the hair and hair follicles should becompletely or partially removed to reduce inflammatory responses invivo. Raw tissue can be partially dehaired by cutting, washing,scrubbing or other means. Further processes for more complete dehairinguse chemicals (for example, depilatory substances) to remove animalhair. Various depilatory substances are contemplated, for example, butnot limited to thioglycolate or sodium sulfide (NaS). Caustic (basic)solutions are also known to remove hair, along with other agents.Potentially suitable bases can be, but are not limited to, sodiumhydroxide (NaOH), potassium hydroxide, lithium hydroxide, Nalco, Ca(OH)₂or NO_(H)O. In some embodiments, the base is NaOH. In some embodiments,the base is used with another depilatory agent, either together or insubsequent steps. Concentrations of less than 5% by weight of NaS arecontemplated; in one embodiment, 1% by weight is used. Concentrations of0.5M or less of NaOH are contemplated; in one embodiment 0.1M NaOH isused. In certain embodiments, NaS and NaOH are used together in a singlestep. In certain embodiments an alkaline solution containing sodiumsulfide having any pH greater than 7 is contemplated, optionally analkaline solution containing sodium sulfide having any pH greater than7.5, optionally an alkaline solution containing sodium sulfide havingany pH greater than 8, optionally an alkaline solution containing sodiumsulfide having any pH greater than 9, optionally an alkaline solutioncontaining sodium sulfide having any pH greater than 10, optionally analkaline solution containing sodium sulfide having any pH greater than11, optionally an alkaline solution containing sodium sulfide having anypH greater than 12. In certain embodiments, solutions which have a pH ofat least 10 and alternatively at least 12 are preferred.

In some embodiments, an aqueous solution of sodium sulfide is 0.01 to 10wt-%, alternatively 0.1 to 5 weight-%, alternatively 0.5 to 3 Wt-%,alternatively 0.75 to 1.5 wt-%, and alternatively about 1 wt-% of sodiumsulfide.

To adjust the above-mentioned alkaline pH-value, it is providedaccording to an embodiment of the present invention that an aqueoussolution is used which in addition to sodium sulfide contain from 0.001to 0.5 M sodium hydroxide, alternatively from 0.005 to 0.4 M sodiumhydroxide, alternatively from 0.01 to 0.3 M sodium hydroxide,alternatively 0.05 to 0.2 M sodium hydroxide, alternatively, 0.075 to0.125 M sodium hydroxide, and alternatively from about 0.1 M sodiumhydroxide.

Instead of sodium hydroxide, other means for setting the pH values canbe used, such as any alkali metal hydroxide or alkaline earth metalhydroxide such as potassium hydroxide, lithium hydroxide, magnesiumhydroxide, calcium hydroxide, ammonia and the like.

It is contemplated that the sodium sulfide solution is used in asufficiently high amount and for a sufficiently long time, so that thedermis is loosened and bristles and hairs are removed almost completely.In some embodiments, the dermis is treated for 1 to 48 hours,alternatively for 5 to 36 hours, alternatively for 12 to 24 hours andalternatively for 14 to 20 hours with an aqueous sodium sulfidecontaining solution. In a specific embodiment, dermis is treated for 16hours with an aqueous sodium sulfide containing solution.

Also, the sodium sulfide-containing aqueous solution volume may be5-fold to 15-fold relative to the volume of the dermis. Alternatively,6-fold to 14-fold volume, alternatively 7-fold to 13-fold volume,alternatively 8-fold to 12-fold volume, alternatively 9 to 11-foldvolume and in certain particular embodiments, approximately 10 times thevolume of solution to the volume of the dermis can be used.

In some embodiments, the treatment with the solution containing sodiumsulfide, may be carried out one or more times in a row, and the dermistreated with water and/or rinsed between the individual treatment stepswith the sodium sulfide-containing solution. In some embodiments only asingle treatment of the dermis with the sodium sulfide-containingsolution is performed.

Treatment with the sodium sulfide solution or any other treatment stepsmay be carried out in a rotating drum. Thereby a uniform and completeincubation of the dermis is achieved with the sodium sulfide or othertreatments. In some embodiments all treatments are carried out in thesame or a similar container, such as a drum. In other embodiments somesteps may be carried out in a different container. In some embodiments acontainer other than a drum may be used.

For example, the rotating drum may be mounted horizontally, and rotateabout its axis. Conveniently, on the drum may be provided connections,through which the process solution feeds into the drum and can beremoved from the drum. Also on the drum connections may be provided asrequired to set a higher or lower pressure. Moreover, in someembodiments the drum is designed with double walls, so that the interiorcavity of the drum can be heated. Finally, in other embodiments, thedrum is designed so that its rotation speed is electronicallycontrolled.

Oxidizing sterilants that may be used in the present processes includeperoxides, oxides, hypochlorites, percarboxylic acids, and ozone. Apreferred peroxide for use in the present processes is hydrogenperoxide. The oxidizing sterilant may be provided in a solution ormixture, with preferred concentration ranging from about 1% to about 15%percent by weight (weight %). In some embodiments, the concentration ofhydrogen peroxide is 3%, other embodiments may use 2%, other embodimentsmay use 4%.

Additionally an osmotic gradient is used during the process, whichgenerates flow in and out of the tissue. This is also referred to asosmotic cycling (transition from hypotonic to hypertonic solutions). Asa non-limiting example, cleaning solutions can be cycled from DI water,to saline and back to DI water.

Saline or salt solutions used in the process include but are not limitedto organic and inorganic salt solutions. For reasons such as easyavailability and cost, the alkali metal chlorides and alkaline earthmetal, in particular sodium chloride, are suitable. Examples ofinorganic salts are well known and include, but are not limited to,NaCl, NaF, NaBr, KCI, KF, and KBr. Examples of organic salts include butare not limited to sodium acetate (CH₃COONa), potassium citrate(C₆H₅K₃O₇), ammonium acetate (NH₄+CH₃COO—), sodium lactate (NaC₃H₅O₃) orother salts resulting from the reaction product of an organic acid andan inorganic base. For reasons such as easy availability and cost, thealkali metal chlorides and alkaline earth metals, in particular, sodiumchloride (NaCl), potassium chloride, lithium chloride, magnesiumchloride or calcium chloride can be used. Specific embodiments mayutilize sodium chloride (NaCl).

Concentrations of salinity (or salt concentrations) contemplated for userange from about 0.6% to 35%. Specific embodiments may utilize about 26%NaCl, alternatively about 1 to 50 weight-% NaCl, alternatively about 2to 20 weight-% NaCl, alternatively about 5 to 15 weight-% NaCl. Otherspecific embodiments use 10% NaCl. Saline (salt) treatments mayalternate with water treatments or DI water treatments one or more timesin certain embodiments, alternatively four times, alternatively fivetimes, alternatively three times alternating between water and saline tocreate an osmotic gradient. These saline treatments in some embodimentsmay last 1 to 48 hours, alternatively 5 to 36 hours, alternatively 12 to24 hours, alternatively 14 to 20 hours, or about 16 hours.

Solvent dehydration is used in the process, preferably near the end ofthe process. The tissue is preferably dehydrated with an organic solventwhich can be mixed with water. Water contents in the solvent mixturerange from about 50% to 0%. Methanol, ethanol, propanol, isopropanol,acetone, methyl ethyl ketone or their mixtures can be used. Specificembodiments may utilize acetone which can be mixed with water. Solventdehydration is achieved, in some embodiments, with an increasinggradient in solvent, for example starting at a lower concentration andfinishing at a higher concentration. In some embodiments, the amount ofacetone is about 65-85 percent, which may vary over certain phases ofthe process. In some embodiments, the water/acetone ratio is 50/50,other embodiments may use 40/60, other embodiments may use 30/70, otherembodiments may use 20/80, other embodiments may use 10/90. In somesteps, 100% acetone is utilized. This can be conducted in an open orclosed bath and racks and trays can be used to hold the tissue, with orwithout shaking. An acetone gradient allows for gentle water removal.Without a gradient (that is, if tissue is immediately placed in 100%acetone), tissue can be harder and not as flexible. Tolerances for theactual concentrations are wide (65-75% and 85-95%) in the first twosteps, then 100% acetone in a final step.

Oven drying is also contemplated, preferably as a last step in theprocess. Appropriate temperatures are used to allow for complete dryingwithout damage to tissue. In some embodiments, an about 37° C. oven isused; in other embodiments an about 35° C. oven. In some embodiments theoven is a circulating-air drying oven. Tissue is dried until desireddryness level is achieved; in some embodiments, this drying is conductedfor about 24 hours, in other embodiments drying is conducted for about16, 18, 20, 22 hours or more. Racks and trays can be used to hold thetissue. In some embodiments, a convection oven with a laminar flow of0.2 m/s is used and drying takes place for at least 24 h at 35° C.

In some embodiments all processing of the tissue is conducted at atemperature of no more than about 50° C., alternatively no more thanabout 45° C., alternatively no more than about 42° C., alternatively nomore than about 40° C., alternatively no more than about 38° C.,alternatively no more than about 36° C., alternatively no more thanabout 35° C., alternatively no more than about 34° C., alternatively nomore than about 32° C.

The cleaning agents of these processes can be contacted with the implantfor a suitable time period in various phases, for example from about 15min to about 30 min, to about 1 hour, for about 1, 2 or 3 hours, or forlonger periods such as about 12 hours, about 16 hours, about 24 hours,about 36 hours or about 48 hours. Phases may be repeated several times,as necessary for complete processing and several steps can be includedwithin one phase (e.g. several rinses included within one phase). Thenumber of phases used depends on the overall process and can includerinsing steps, storing or soaking steps, drying steps, etc. Ten or morephases are contemplated, in some embodiments 15 or more phases are used,in some embodiments 20 or more phases are used, in some embodiments 25or more phases are used, in some embodiments 26 or more phases are used.

Certain procedures are used in initially providing the dermal tissue forprocessing. The procedures include, but are not limited to, cleaning theskin of slaughtered pigs with detergent-containing water at atemperature of not more than about 40° C.; removing the skin from theback portion of the pig; removing the bristles from the skin providedabove and removal of the subcutaneous tissue and the epidermis from thedermis using a splitting machine.

Detergents to be used for initial cleaning of the skin can be a nonionicdetergent, an anionic detergent or both. Nonionic detergentscontemplated for use in the process of the application include, but arenot limited to, an alcohol ethoxylate, an alkylphenol ethoxylate, analkyl polyglycoside, a polyoxyethylene ether, a polyoxyethylenesorbitan, or any of the Triton®, Tween® or Brij® series of detergents(e.g. Triton® X-100). Anionic detergents contemplated for use in theprocess of the application include, but are not limited to, an alkylbenzenesulfonate, an alkyl sulfonate, an alkyl phosphate or an alkylsulfate, such as the sodium salts of dodecyl sulfate, myristyl sulfate,cetyl sulfate, steryl sulfate and oleyl sulfate (e.g. sodium dodecylsulfate (SOS; also called SLS)). In some embodiments, sodium dodecylsulfate (SOS) is preferred.

Specifically in reference to dermal tissue, the tissue is split bymechanical means prior to processing. Only the inferior region of thetissue is utilized for processing, the top of the midsection of thetissue is contemplated. In one embodiment, the raw dermal tissue isrecovered from pig (porcine) hide, preferably from specific locationsthat have improved mechanical properties. The raw tissue is dehaired anddefatted by cutting away the undesired portions. The tissue is thenpulled through a splitter that has a rotating blade. The tissue ispassed through several times until a desired thickness is reached. Insome embodiments thicknesses of less than 2 mm or less than about 2.0 mmis desired, preferably 1.5 mm or about 1.5 mm, alternatively 1.0 mm orabout 1.0 mm, alternatively less than 1.0 mm or less than about 1.0 mm.Tolerances for thickness are about 0.2 mm. After splitting the tissue istreated with the cleaning agents, as described above.

In certain embodiments, the splitter may have a bandsaw blade. Incertain embodiments the splitter may have an endless knife, similar to abandsaw blade having no teeth. In some embodiments the blade may bemounted horizontally, alternatively vertically, alternatively at anangle to the vertical or to the horizontal which could be 15° or 30° or45° or 60° or more. In some embodiments the tissue may be delivered tothe splitter blade by one or more rollers to be split along the crosssectional area. In certain embodiments, the tolerance for the thicknessmay be 0.5 mm, alternatively 0.4 mm, alternatively 0.3 mm, alternatively0.1 mm, alternatively 0.05 mm.

One or more processing steps may be conducted in a mixer or drum whichfunctions to create turbulent flow of the chemicals. Suitable mixerstumble the tissue in a chamber, optionally with baffles to furtherimprove turbulent flow. In one embodiment, a rolling drum mixer isutilized. The filling and emptying of the rolling drum mixer can beconducted with a pump and with venting, and may be made more efficientor more rapid by the use of valves and connectors on the drum, supplylines from the chemical sources, or automated control of chemical flow,filling draining and rolling or mixing. In some embodiments the mixingmay be carried out continuously throughout all, most, or some portion ofeach processing step. In certain embodiments the mixing is continuousthroughout the majority of time, alternatively about half the time,alternatively about three-quarters of the time, alternatively about 80%of the time, alternatively about 90% of the time for at least one step,alternatively for two consecutive steps, alternatively for threeconsecutive steps, alternatively for four or more consecutive steps inthe process.

In certain embodiments, the processing may occur in an open or closedvessel. For example, certain aspects of the present application may becarried out in whole or in part within a metal, glass or polymericbeaker or basin with a smooth rim for ease of handling tissues andpouring chemicals into and out of the vessel. Certain aspects of theapplication may also be practiced using a vessel with a threaded orcompression fit lid which screws or snaps on to provide closure toprevent spills or contamination during processing, handling, transportor storage of the tissue.

For xenograft tissue, crosslinking can be used to reduce immunogenicityand strengthen tissue. Chemical agents (such as, for example,glutaraldehyde) used for crosslinking can have deleterious side effects,however. Although crosslinking of the tissue is contemplated, in certainembodiments the tissue is preferably not crosslinked. In certainembodiments, the porcine dermis implant is comprised of non-crosslinkedporcine dermis, which acts as a scaffold that allows forneovascularization and reincorporation with a patient's own tissue.

Additionally additional osteoconductive or biologically active materialssuch as demineralized bone matrix (DBM), mineralized bone matrix,cortical cancellous chips (CCC), crushed cancellous chips, tricalciumphosphate, hydroxyapatite, biphasic calcium phosphate, muscle fibers,collagen fibers, growth factors, antibiotics, cells, or other additivescan be utilized in the grafts of the present application. These could beadded to the grafts by infusion or used as part of a layered matrix.

After acellular tissue is created through the process of thisapplication as described above, the tissue can be shaped into desiredgraft configurations. The shaping can be created via punching (alsocalled stamping) or cutting, either by an automated or manual process.Stamping/punching can be performed with a preshaped dye to createdesired shapes. Graft shaping can be performed either wet or dry. Ifgrafts are dry, then they can be rehydrated (e.g. with water) before orafter shaping. In some embodiments, grafts are dried after processingvia solvent dehydration, further dried in an oven, rehydrated with waterand then shaped via punching/stamping. Rehydration can be performed instainless steel or plastic tubs, with or without sealing lids. In someembodiments rehydration is performed for 30 min, in other embodimentsrehydration is performed for 45 min, 60 min or more.

Dermis grafts can be provided in several shapes. The shape ispredetermined and is based on shapes and sizes most commonly needed forsurgeries (e.g. hernia surgery). Preferred shapes or configurations area square, rectangle, strip, circle, triangle or oval. Irregular,asymmetric, symmetric, geometric, custom, anatomical, and patternedshapes are also contemplated. For shapes such as rectangles, squares orstrips, corners of the grafts can be rounded for ease in use in surgery.Rounded corners (radii) of 20 mm or less are contemplated. In oneembodiment the rounded corners (radius) is 15 mm.

In some embodiments, shapes such as squares and rectangles arecontemplated. In some embodiments, the grafts have dimensions betweenabout 50 mm and 500 mm. In other embodiments, the grafts have dimensionsof about 400 mm, 350 mm, 300 mm, 250 mm, 240 mm, 230 mm, 220 mm, 210 mm,200 mm, 180 mm, 170 mm, 160 mm, 150 mm, 140 mm, 130 mm, 120 mm, 110 mm,100 mm, 90 mm, 80 mm, 70 mm, 60 mm, or 50 mm. Tolerances for length andwidth can be 3%, in some embodiments the tolerance is not less than 5mm.

Optionally the implants (grafts) are packaged and further sterilized.Packaging can include pouches, bags or other sealable articles. Innerand outer pouches can be used. Grafts can be stored (e.g. at ≤20° C.,alternatively at ≤4° C.) before, during or after further sterilization.Further sterilization can include sterilizing with gamma rays(gamma-irradiation) with a minimum dosage of about 15 kilogray (kGy). Inother embodiments, irradiation is carried out with a dosage of about 20kGy.

In certain embodiments, the grafts are stored hydrated and ready-to-use(RTU). Hydration can be performed with biocompatible fluids, for example(but not limited to) water or saline. In some embodiments, the graftsare sealed in a package, and ready-to-use upon removal from the package.Sealing the package using moisture resistant packaging allows forextended storage in a ready to use form. Such packaging allows for thegraft to remain in a hydrated form. The grafts can be sealed, forexample, in a pouch, and the pouches can be sterilized, such as by lowdose gamma irradiation. In certain embodiments, the porcine dermisimplant is comprised of non-crosslinked porcine dermis, designed to actas a scaffold that allows for neovascularization and reincorporationwith the patient's own tissue.

The present application in some embodiments comprises a soft tissueimplant, useful for soft tissue repair procedures such as for herniarepair, designed from xenograft tissue that would take advantage of hightissue availability and animal domestication to be able to control thesource tissue material. Xenografts can be porcine, ovine, ratite,equine, caiman, bovine or any other suitable animal source. In certainembodiments the xenografts are preferably porcine.

For grafts to be utilized in hernia repair and other such surgeries,mechanical characterization requirements of load at failure at or abovenominally 39N and 10±3.4 N/mm² were pre-determined based on literaturereview [Hollinsky]. Unprocessed porcine dermis does not have theserequired mechanical properties. The goal was to develop a process fortreating the xenograft tissue to obtain the required properties socreate tissue suitable as a graft for implantation. Testing showed thatinitial versions of the processed grafts showed variability inbiomechanical strength and prompted an investigation into betterunderstanding the variability in relation to the anatomical region ofthe dermis of the samples.

It was determined that the mechanical properties of porcine dermis varyaccording to the anatomical location on the hide that the samples aretaken from. FIG. 1 shows the anatomical locations studied.

FIG. 1 shows the regions of porcine tissue for evaluation. The porcinedermis was divided into eight pieces and the pieces were labeled byanatomical location using an alphanumeric identification system. Letterdesignations from A to D were used to identify the cranial (A) to thecaudal (D) regions. A numerical identifier was used for the dorsal (1)to the ventral (2) regions. Mechanical property testing showed that thecranial (region A) and ventral tissues (region 2) showed the weakestbiomechanical properties. The B1 and D1 regions showed uni-axialmechanical properties that meet the nominal requirements of load atfailure at or above 39 N and 10 N/mm².

EXAMPLES Example 1: Processing Porcine Dermis

Porcine dermis samples were processed for evaluation. The process forpreparing these samples involves hair removal, hypo/hyperosmotic soaks,hydrogen peroxide, solvent dehydration, and oven drying.

Specific processing of these grafts included storage in 26% NaCl, hairremoval with a sodium sulfide/sodium hydroxide (H₂0₂) mixture, multiplehypo/hyperosmotic soaks (utilizing NaCl solutions and water), hydrogenperoxide treatment, and then solvent dehydration using acetone, and ovendrying at 37° C.

Histological examination of these processed porcine dermal samples wasperformed, see FIGS. 2A and 2B. FIG. 2A shows H&E stained tissue with50× magnification and frozen cryostat sectioning. FIG. 2B shows AlcianBlue stained tissue with 100× magnification and frozen cryostatsectioning.

Histological evaluation of the Hematoxylin and eosin (H&E) sections ofthe processed porcine dermal tissue showed an open porous matrix, seeFIG. 2A. This architecture was consistent throughout the tissue. Thetissue showed no significant cellular debris, no remnant hair follicles,and no notable adipose throughout the porcine dermal matrix. Thehistoarchitecture of the native dermal matrix is maintained. Themechanical evaluations of the processed porcine dermis meet thespecified requirements of 39N and 10 N/mm² loads at failure.

Example 2: Processing Porcine Dermis

A freshly slaughtered pig was washed in 40° C. warm water, with 0.1 to 1wt-% sodium dodecyl sulfate (SOS), for superficial cleaning, before itwas skinned. Of the entire skin, the portions of the side of the abdomenwere removed and discarded, and only the skin of the back with an areaof approximately 800×1200 mm processed.

Then bristles were removed mechanically from the pig skin with a bladebefore the skin was cut into approximately 400×600 mm sized pieces.After the skin has been introduced with the fat side up into a splitterwith two driven wheels and horizontally mounted endless knives with agap size of 2 mm to separate the excess fat and binder componentsincluding the hair roots. Subsequently, the gap dimension of thesplitting machine is set to 0.5 mm, and the skin was again passedthrough the splitting machine to cleave the epidermis, leaving behind adermis with a thickness of 1.5 mm.

Then the dermis was treated according to the following treatment regimenin a rotating drum:

Step Action Time (hours) 1 Rinse with water (3x) — 2 store in water 0.53 store in water 0.5 4 treatment with aqueous 1% Sodium sulfide, 16 0.1MNaOH solution 5 Rinse with water (3x) — 6 store in water 1 7 store in10% NaCl 3 8 store in water 1 9 store in 10% NaCl 16 10 Rinse with water(3x) 11 store in water 1 12 store in 10% NaCl 3 13 store in water 1 14store in 10% NaCl 16 15 Rinsing with water (3x) — 16 store in 3%hydrogen peroxide 24 17 store in 3% hydrogen peroxide 24 18 store in 3%hydrogen peroxide 24 19 Rinse with water (3x) — 20 store in 70% acetone2 21 store in 90% acetone 2 22 store in 100% acetone 2 23 store in 100%acetone 16 24 store in 100% acetone 24 25 store in 100% acetone 24 26drying at 35 [deg.] 24

After the chemical treatment, dermis grafts were punched from thedermis. Grafts were oval in shape and had dimensions of 160×250 mm.These were pairwise welded dry in a laminated plastic film, at 35° C.and gamma-irradiated with a dose of 20 kGy. The thus prepared graftsshowed a nearly identical morphology to the native structure.

Example 3: Rat Model Study

Grafts made by a process of the application were studied in a rat model.A sham procedure was used as a control in this study. Sprague-Dawleyrats received a unilateral defect, which was repaired with eitherprimary closure or an implanted graft. This study evaluated in vivocellular infiltration, neovascularization, encapsulation,hematoma/seroma formation, tissue remodeling, and biomechanics at 4- and8-week sacrifice intervals. This study also evaluated un-implanted grafthistology, biomechanics, and tissue degradation properties.

Sprague-Dawley rats were selected based on availability, limited testingexpense, and the immune competency of these rats. Sprague-Dawley ratshave been used in various other studies with processed soft tissuesi.e., human dermis, with positive results. These studies includedbilateral defect models and bilateral subcutaneous models. The lineaalba defect site was used in the current study to mimic existingliterature [1-5].

The decellularization process for the porcine dermis used in this studywas performed with H₂0₂, osmotic treatment (gradient), Sodium Sulfideand NaOH, and Acetone as in Example 1.

Group I was designated as a control group. Group I rats had a shamprocedure performed at the time of surgery, which was repaired byprimary closured. Test group rats received a full-thickness abdominalwall defect, which was repaired with a pre-determined acellular porcinedermal matrix [3-5]. Surgical defects were created approximately 0.5 cmlateral to the midline.

The processed porcine dermal samples were prepared for implant themorning of surgery. Sterile technique was used to section all samplesfor implantation. Samples were sectioned for surgical implant (2×3 cm)in a laminar flow hood. Additional sectioning for mechanical testing,enzymatic degradation, and histology was done non-sterilely. Sterile 50mL centrifuge tubes used for re-packaging samples sectioned forimplantation. Samples were transported to the surgical suite in a polybag closed with a zip-tie. Samples for time zero evaluation were removedfrom the hood and tested the day of surgery.

Un-implanted tissue evaluation was performed. Porcine dermis sampleswere processed, cut-to-size, packaged individually, and terminallysterilized. Samples were shipped at ambient temperatures to thefacility, but temperature during shipment was not tracked. All sampleswere sectioned for implantation and time zero (T=0) testing in a laminarflow hood using aseptic technique the morning of surgery.

Time zero tissue evaluation was performed using histology, tissuethickness measurement, and uni-axial tension testing. This testing wasconducted to evaluate tissue cellularity, extracellular matrixproperties, degradation properties, and biomechanical properties.

Samples for histological evaluation were sectioned at the time of sampleprep for implantation and all extra tissue samples were repackaged andsent for mechanical testing and enzymatic degradation. Samples forhistology were immediately submerged in 10% buffered formalin and sentfor paraffin embedding and sectioning. Samples were sectioned between 5and 10 μm thick, mounted on a histology slide, and stained with theappropriate histology staining procedure. Sections were stained with oneof the following: H&E, Alcian blue, or Verhoeff Van-Gieson staining. Allsamples were evaluated after sectioning and staining with a bright fieldmicroscope.

Enzymatic degradation assay samples were run in duplicate, with threereplications to attain an average of values for each sample. Opticaldensity values were used to calculate mg of degraded collagen per mL ofdigest solution.

Mechanical testing was performed on an Instron (Model #3366). Alluni-axial samples were cut into a dogbone configuration prior totesting. Ball burst samples were cut to 2×2 cm prior to testing. Sampleswere stored in 0.9% normal saline until mechanical testing. Testing datawas used to determine ultimate load at failure and strain.

For surgical implantation, Group I rats, the sham control, received a 2cm incision lateral to the linea alba (craniocaudal). Two 0.3-0.5 cmtransverse incisions, one at the cranial end and one at the caudal end,were created to make a muscle flap. The muscle flap was sutured to theopposing side of the abdominal wall with 5-0 non-resorbable suture usinga simple interrupted pattern and ensuring at least 0.3 cm of overlappingtissue.

Rats in the graft implant groups, received a 1×2 cm full-thicknessdefect lateral to the linea alba. This defect was repaired with apre-determined implanted matrix. Implants measuring 2×3 cm were used torepair the defect in an overlay fashion. The implants were sutureddirectly to the abdominal wall, with a minimum of 0.3 cm of overlapbetween the implant and abdominal wall on each edge of the implant.Implants were sutured with 5-0 non-resorbable suture. All samples weresutured onto the abdominal wall using a simple interrupted pattern; nobuckling was present in any of the samples.

At explant, animals were sacrificed at 4 and 8 weeks after surgery. Atthe time of explant, animals were humanely euthanized. The midline ofeach animal was opened to access the samples. The samples were excisedto include the entire implant or sham repair and a small margin of theabdominal wall. Hematoma/seroma presence was noted at the time ofexplant.

Each sample was explanted with at least 1 cm of abdominal wall on theedge of each implant for mechanical testing sites. All test explantswere photographed with appropriate labeling for identification of testarticles. Each explant was measured after excision from the abdominalwall. Explant samples were sectioned to attain two mechanical testingsamples and one histology sample. The samples for mechanical testingwere sectioned and placed in labeled conical tubes and covered with 0.9%normal saline. Samples were uniaxially tensioned until failure. Samplesfor histological sectioning were collected with at least 0.5 cm ofabdominal wall around the edge of each implant and trimmed to size priorto being submerged into 10% buffered formalin. Samples were sent forsectioning and staining. Samples were embedded in paraffin, sectioned 5μm thick, and stained with Hematoxylin & Eosin. Evaluation ofhistopathology data was used for the evaluation of the biologicalresponse at 4 and 8 weeks in vivo.

Un-implanted samples of processed porcine dermis measured 1.56 to 1.6 cmthroughout the entire material. The pH of the samples was between 6.0and 6.5 directly out of the package or post-hydration. Histologicalevaluation of all porcine dermal matrices showed an intact extracellularmatrix and no definitive cellular debris.

The protocol utilized for enzymatic degradation is designed to degradethe tissue in vitro over a 24 hour period. The tissue samples are soakedin an enzyme solution at 37° C. for 1, 4, 8, and 24 hours. The solutionis filtered to isolate the degraded collagen. The hydroxyproline is thentagged in the solution; this produces various shades of purpleindicating concentration of hydroxyproline in the solution. The opticaldensity is measured by an enzyme-linked immunosorbent assay (ELISA)plate reader. The data collected is reported as the collagen degraded inmg per ml of solution. All samples showed a predictable degradationcurve over the 24-hour evaluation.

Un-implanted (T=O) samples were evaluated via uniaxial tension testingand ball burst mechanical testing. Uni-axial tension testing data wasevaluated with maximum load at failure in Newtons (N) and the load atfailure normalized to cross-sectional area (stress) in megapascals (MPaor N/mm2). Porcine dermis grafts had maximum load at failure of52.04±19.9 N and a maximum stress at failure at 6.03±2.0 MPa. The graftsalso had high burst strengths of 324.59±62.82 N.

Animals were explanted at 4 weeks post-operative and showed someresidual signs of hematoma/seroma and minor adhesions. Animals used forthe sham control showed no signs of adhesions or hematoma/seroma. Therewas no noted presence of neutrophils, mineralization, necrosis, orencapsulation in any of the samples at the 4-week sacrifice interval.The sham control showed no presence of plasma cells, eosinophils,neutrophils, foreign body reaction, or hematoma/seroma at the 4-weeksacrifice interval. The histological evaluation of the 8-week samplesshowed no indication of the presence of hematoma/seroma, mineralization,necrosis, or encapsulation in any of the samples. There was no foreignbody reaction noted in the sham control or in the implants of thecurrent application.

For mechanical testing, the in vivo mechanical samples were sectionedinto cranial and caudal portions. The cranial section had a largerportion of intact abdominal wall compared to caudal sections. Thedog-bone punch was oriented on each sample to maintain the gauge areacentered over the original defect and/or the remaining implanted matrix,while the grip area consisted of abdominal muscle. This ensured theevaluation of the shear properties of the interface strength between theimplant the abdominal wall. The porcine dermis grafts exhibited strengthat 4 weeks of 7.6N and at 8 weeks of 12.3 N.

There were improvements from 4 to 8 weeks in shear mechanical integrityin all grafts. The grafts had approximately 62% increase in interfacestrength, and the sham had approximately 40% increase in interfacestrength from 4 to 8 weeks.

All implanted tissues showed little to no trace of residual cellulardebris in the histological sections, which was confirmed by themild/moderate inflammatory process noted in these samples. There was nonoted presence of eosinophils, a cell type indicative of an allergicreaction. The largest differences noted in cell infiltration were thelymphocytes and macrophages. The fibroblast scores increased in allgroups from 4 to 8 weeks, which is a naturally occurring process duringwound healing. The neovascularization and fibrosis scores were asexpected. The implants of the application did not exhibit a foreign bodyreaction at 8 weeks.

The samples showed acceptable ultimate load and stress at failure andbiaxial burst strength. The un-implanted biomechanics, while usefulinformation, are not necessarily indicative of in vivo mechanicalproperties during the wound healing process. Samples showed asignificant increase in shear load from 4 to 8 weeks, which isconsistent with the collagen deposition and crosslinking process thatoccurs after surgical intervention.

The biomechanics and inflammatory responses noted in this study showthat the porcine dermis implant samples showed good incorporation, amild to moderate inflammatory response, neovascularization, noencapsulation, no persistent hematoma, and no persistent foreign bodyreaction. The biomechanics and biological responses were withinacceptable values for dermal meshes. Overall, the results of this studywere highly positive indicators that porcine dermis processed using themethods of the present application is rendered sufficiently lessimmunogenic to prevent acute rejection of the implant.

All patents, test procedures, and other documents cited herein,including priority documents, are fully incorporated by reference to theextent such disclosure is not inconsistent with this application and forall jurisdictions in which such incorporation is permitted.

While the application has been described with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the application. In addition, manymodifications may be made to adapt a particular situation or material tothe teachings of the application without departing from its scope.Therefore, it is intended that the application not be limited to theparticular embodiment disclosed, but that the application will includeall embodiments falling within the scope of the appended claims.

1-12. (canceled)
 13. A method for the preparation of a graft from bovinedermis comprising: providing a bovine dermis having a native dermalmatrix; treating said dermis with an aqueous, alkaline sodium sulfidesolution for at least 1 hour, wherein said solution comprises 0.01% to1.5% by weight of sodium sulfide and 0.001 M to 0.5 M sodium hydroxideand said solution has a pH greater than 10; once or several times,treating the dermis with a saline solution; once or several times,treating the dermis with an aqueous hydrogen peroxide solution;dehydrating the dermis via solvent dehydration that starts with a lowerconcentration of solvent and finishes with a higher concentration ofsolvent; and maintaining a histoarchitecture of the native dermal matrixwherein the graft is non-crosslinked.
 14. The method of claim 13,wherein the dermis is treated with the aqueous sodium sulfide solutionfor 1 to 48 hours. 15-18. (canceled)
 19. The method of claim 13, whereintreating the dermis with a saline solution comprises treating two to tentimes with the saline solution, wherein the saline solution comprises 1%to 50% by weight of salt, and wherein the salt is an alkali metal oralkaline earth metal, selected from the group consisting of sodiumchloride, potassium chloride, lithium chloride, magnesium chloride, andcalcium chloride.
 20. The method of claim 13, wherein treating thedermis with the aqueous hydrogen peroxide solution comprises treatingtwo to five times with the aqueous hydrogen peroxide solution, andwherein the aqueous hydrogen peroxide solution comprises 1% to 10% byweight of hydrogen peroxide.
 21. The method of claim 13, whereindehydrating the dermis the dermis comprises dehydrating two to ten timeswith an organic solvent selected from the group consisting of acetone,methanol, ethanol, and isopropyl alcohol.
 22. The method of claim 13,further comprising a rehydrating step the graft.
 23. The method of claim13, further comprising punching or stamping grafts of a desired shapefrom the animal dermis.